Ulrich Hintze

Dead but Highly Dynamic – The Stratum corneum Is Divided into Three Hydration Zones

Topically applied water exerts mechanical stress on individual corneocytes as well as on the whole stratum corneum (SC), resulting in an alteration of barrier function. In this study we used complete skin biopsies and showed that the SC reacts to water stress as a highly optimized and well-regulated structure against osmotic changes. Following a relatively new cryo-processing protocol for cryo-SEM, it is possible to reliably maintain and investigate the hydrated state of the SC and individual corneocytes after treatment with solutions of different ionic strength. Treatment with distilled water results in swelling of SC cells together with formation of massive water inclusions between adjacent cell layers. Treatment with 5–20% NaCl reveals three different hydration zones within the SC: Corneocytes near the live-dead transition zone can swell to nearly double their thickness. The second zone is the most compact, as the corneocytes here show the smallest thickness variation with all treatments. Within the outermost zone, again a massive swelling and loosening of intracellular filament packing can be observed. We therefore conclude that the SC itself is subdivided into three functional zones with individual water penetration and binding potentials. Since the second zone remains nearly unaffected by water stress, we propose that this zone hosts the functional SC barrier.

From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging.

Structural investigation of tissue biopsies requires the coupling of optimal structural sample preservation with detailed information collected at the light and electron microscopic level. Unfortunately, although cryo‐immobilization by high‐pressure freezing provides the best structural preservation, it is used routinely only for electron microscopic (EM) investigations, whereas for light microscopy chemical fixation protocols have been established. These chemically invasive fixation protocols have the drawback of introducing unpredictable fixation artefacts. Therefore, comparative histopathological (i.e. light microscopic) and ultrastructural (i.e. EM) results are usually obtained from parallel samples that have not been prepared identically and never by examining exactly the same features in exactly the same, optimally preserved sample. Finally, finding an area of interest for EM investigation within a complex tissue is like searching for a needle in a haystack. To overcome these handicaps, we modified the well‐established freeze‐substitution technique (FS) to allow us to investigate resin‐embedded cryo‐immobilized tissue by confocal laser scanning microscopy (CLSM) prior to EM examination. Thus (1) selected cells throughout the whole tissue block can be depicted by CLSM and subsequently (2) selectively prepared by targeted sectioning for follow‐up investigation of the identical structure by transmission electron microscopy. This is facilitated by the addition of specific fluorescent dyes during the first FS exchange step. Selective binding properties of various dyes to different cellular structures allow a direct histological description of the tissue at the light microscope level. After embedding and preparation of a blockface, the specimen can first be examined by CLSM. For areas of interest, the depth in the resin block is determined followed by removal of the tissue lying above. Then, the cell layer can be cut into a series of ultrathin sections and examined by EM for determination of the subcellular and nanostructural organization.

Comparative investigation of human stratum corneum ceramides

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, without further analysis, some uncertainty remains about the molecular composition of lipids represented by every TLC band of an unknown sample. We therefore analyzed each ceramide band further by subjecting the isolated lipids to a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or IC/MS). IC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociation experiments revealed that ceramide band 4 contained not only N-(ω-OH-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(α-OH-acyl)-sphingosine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results demonstrate the benefit of LC/MS analysis for selective identification of human SC ceramides. Moreover, the combination of HPTLC for pre-separation and LC/MS for identification of lipids is an even more powerful tool for detailed ceramide analysis.

QUANTITATIVE DETERMINATION OF NATIVE AND METHYLATED CYCLODEXTRINS BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

Abstract A method for the quantitative analysis of cyclodextrins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is described. To decouple the quantitative approach from the extreme dependence of the absolute ion signal on the experimental parameters of the MALDI technique, the method relies on the normalization of analyte molecular ion signals relative to internal reference molecular ion signals. For good crystal homogeneity and shot-to-shot reproducibility a new method of sample preparation using 4-hydroxy-α-cyanocinnamic acid as a matrix was developed. Linear calibration curves ( r 2 = 0.998) were obtained for both permethylated cyclodextrins with homologous standards and β-cyclodextrin using maltohexaose as internal standard. Furthermore, tests on different 2,3- and 2,6-di- O -methylated cyclodextrins were made. An excellent shot-to-shot reproducibility allowed the analysis of byproducts using the molecular ion signal of the product as internal standard. In each instance, the results demonstrate that MALDI is a viable approach for quantification of low-molecular-mass analytes.

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy.

Thin cross‐sections of human hairs were investigated by scanning near‐field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross‐sections of hairs.

Rapid quantification of iodopropynyl butylcarbamate as the preservative in cosmetic formulations using high-performance liquid chromatography-electrospray mass spectrometry

A simple, rapid and reproducible method for identification and quantification of iodopropynyl butylcarbamate (IPBC) in different cosmetic formulations is presented. The determination was carried out using a high-performance liquid chromatography (HPLC) procedure on a reversed phase column coupled to a single quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the positive selected ion-monitoring mode. In methanol/water extracts from different cosmetic formulations a detection limit between 50 and 100 ng/g could be achieved. A routine analytical procedure could be set up with good quantification reliability (relative standard deviation between 0.9 and 2.9%).